Historically, the production of reactive oxygen species (ROS) by phagocytes was mainly studied in the context of bacterial killing. Then, it was established that ROS controls normal physiologic signaling termed as redox-signaling in many cell types. The recent emerging concept is- macrophages that are far less potent than neutrophils at producing ROS, acquire their activated microbicidal phenotype through the production of proinflammatory cytokines which is orchestrated by redox-signaling. On the flip side, bacterial antioxidant defenses neutralize macrophage-derived ROS to survive in macrophages and these mechanisms have been well established. However, the impact of ROS neutralization by bacterial antioxidants on redox-signaling pathways, pro-inflammatory cytokine production, and innate immune responses remain understudied. The goal of the proposed research is to establish a novel role for bacterial antioxidants in the modulation of host's innate immune response using a category A select agent, Francisella tularensis (Ft) SchuS4 as a model. We are particularly interested in investigating the mechanisms by which Ft suppresses host's innate immune defenses. Our extensive work in the pursuit of understanding the virulence mechanisms of Ft lays a very solid foundation for the proposed research program. We have demonstrated that Ft SchuS4 possesses robust antioxidant defense mechanisms which render it extremely resistant to oxidants. By generating point and multiple gene deletion mutants of a close surrogate of Ft SchuS4- the live vaccine strain (LVS), we have established that Ft antioxidants are important virulence factors and play an essential role in the suppression of kinase signaling and macrophage activation. In this application, we wish to dig deeper to expose the link between efficient ROS scavenging capacity of Ft SchuS4 to its ability to cause innate immune suppression. Our published and preliminary studies provide compelling evidence in support of our hypothesis that Ft antioxidants suppress innate immune responses by inhibiting the activation of redox-sensitive signaling components and production of pro-inflammatory cytokines. The following specific aims are proposed: Aim 1: will investigate how antioxidants of Ft SchuS4 subvert innate immune function by modulating macrophage redox-environment. Aim 2: will investigate the impact of Ft antioxidant defenses immediately upon uptake, on intracellular macrophage ROS-dependent signaling cascades, while Aim 3 will establish the mechanism of Ft antioxidant mediated subversion of cytosolic redox-sensitive nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling. The long-term objective of this application is to build a strong platform for the development of redox- based broad-spectrum therapeutic/prophylactic approaches. The concept that bacterial antioxidants in addition to their established role as scavengers of ROS/RNS can subvert host's innate immune response is highly innovative and may truly uncover a novel and conserved mechanism of bacterial pathogenesis.